Nitric oxide (NO) is a small free radical that is synthesized from L- arginine by nitric oxide synthase. NO is increasingly recognized as a major control point in critical physiological and pathophysiological processes. This highly reactive molecule may participate in acute and chronic lung injury. The lung contains various cells known to produce NO (e.g., endothelial, epithelial, and neuronal cells as well as cells that may temporarily reside in the alveolar space such as macrophages, lymphocytes, neutrophils, platelets, and mast cells). Strategically placed in the circulation, the lung serves as an immunologic filter; it is the first line of defense against exogenous NO from air pollutants and cigarette smoke. The lung is an oxygen-rich environment, allowing for the reaction of NO with oxygen-derived molecules, generating highly reactive compounds. Defining the molecular mechanisms involved in the regulation of NO synthesis may help elucidate the role of NO in the pathogenesis of lung diseases. Inducible nitric oxide synthase (iNOS), the critical enzyme responsible for the enhanced synthesis of NO in inflammatory states, is widely expressed in lung cells. To delineate regulatory roles of the 5'-untranslated region (5'-UTR) of the human iNOS, the transcription initiation sites and structure of the 5'-UTR were examined. Freshly isolated human alveolar macrophages, bronchial epithelial cells and several types of cultured cells were evaluated following stimulation with cytokines (interferon-gamma, interlukin- 1beta, tumor necrosis factor-alpha, and interlukin-6). The mRNA was analyzed by reverse transcription-polymerase chain reaction, Northern analysis, and 5'- rapid amplification of cDNA ends. Despite the presence of a TATA box in the promoter region, multiple transcription initiation sites were observed, some extending several hundred base pairs upstream from the main TATA-directed initiation site. Alternative splicing in 5'- UTR of human iNOS mRNA resulted in further diversity. The TATA- independent iNOS mRNA transcripts were up-regulated by cytokines. The long and complex 5'-UTRs contain eight partially overlapping open reading frames upstream of the putative ATG of human iNOS, which may have an important role in translational regulation of human iNOS mRNA, and thus the formation of NOS protein, and the generation of NO.